Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia

doi:10.1016/S0006-8993(97)00089-9

Brain Research

Volume 755, Issue 1, April 1997, Pages 36-46

https://doi.org/10.1016/S0006-8993(97)00089-9 Get rights and content

Abstract

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre-and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.

Introduction

In cerebral ischemia the reduction in the supply of glucose and oxygen to the brain leads to a complex cascade of cellular events, resulting in both acute and delayed neuronal death [49,50]. Many aspects of ischaemic neurodegeneration have been demonstrated in animal models, however, these represent a complex environment in which to dissect the cellular and molecular mechanisms involved in ischaemic neurodegeneration. Cell culture systems, while further from the in vivo situation represent a more defined microenvironment where the presence of a vascular compartment and changes in temperature, for example, are not confounding variables. Most studies to date have used mixed neuronal and glial cultures to investigate the effects of oxygen and/or glucose deprivation [20,32,46,57]. Regional organization and neuronal connectivity is lost in most mixed astrocyte/neuronal cultures, however, in organotypic cultures cell stoichiometry and regional connectivity are substantially retained [17,52]. In addition hippocampal slice cultures retain a similar glutamate receptor population compared to that found in adult animals [1]. As glutamate-mediated mechanisms may play an integral role in the generation of ischaemic damage this further supports the use of organotypic slice cultures for the investigation of neuronal damage following ischaemia.

Section snippets

Materials and methods

Organotypic hippocampal slice cultures were prepared according to the method described by Stoppini et al. [52]. Eight to 10-day-old Wistar rat pups were killed by decapitation and the hippocampi rapidly dissected out. 400 μm transverse sections were prepared using a McIllwain tissue chopper and placed into ice cold Geys balanced salt solution (supplemented with 5 mg/ml glucose and 1.5% Fun-gizone, GIBCO/BRL, Renfrewshire, UK). Cultures were placed onto semiporous membranes (Millipore, Watford,

Results

After 2 weeks in vitro, more than 95% of cultures were viable (as determined by lack of PI fluorescence) and maintained an organotypic organization with clearly defined neuronal cell layers (as confirmed by thionin staining). The neurones within the pyramidal and dentate gyrus cell layers had large, spherical, weakly-staining nuclei surrounded by strongly-staining cytoplasm. Immunostain-ing for GFAP demonstrated the presence of a well defined glial matrix within the culture (Fig. 1).

Discussion

We observed no neuronal damage in cultures deprived of glucose for 24 h. Using dissociated cultures of either hippocampal or cortical neurones many groups have shown that a significant proportion of neurones are lost 24 h after a prolonged period of glucose deprivation (4–8 h [14,32,33]; 15–24 h [6,7,27,34]). In contrast, other groups [38,51] have found that in similar culture systems long-term glucose-deprivation alone does not reduce neuronal viability. During the preparation of dissociated

Acknowledgements

This work was supported by a grant from The Wessex Medical School Trust.

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Research reportNeuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

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Research report
Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia